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- PDB-6xly: CRYOEM STRUCTURE OF MYCOBACTERIUM TUBERCULOSIS ZINC METALLOPROTEA... -

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Basic information

Entry
Database: PDB / ID: 6xly
TitleCRYOEM STRUCTURE OF MYCOBACTERIUM TUBERCULOSIS ZINC METALLOPROTEASE ZMP1 IN OPEN STATE
ComponentsProbable zinc metalloprotease Zmp1
KeywordsHYDROLASE / Open state
Function / homology
Function and homology information


protein processing / metalloendopeptidase activity / metal ion binding / plasma membrane
Similarity search - Function
Neprilysin-like (M13) protease domain profile. / Peptidase M13 / Peptidase M13, N-terminal domain / Peptidase M13, C-terminal domain / Peptidase M13, domain 2 / Peptidase family M13 / Peptidase family M13 / Metallopeptidase, catalytic domain superfamily
Similarity search - Domain/homology
Probable zinc metalloprotease Zmp1
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLiang, W.G. / Zhao, M. / Tang, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)R01 GM121964 United States
CitationJournal: Structure / Year: 2021
Title: Structural analysis of Mycobacterium tuberculosis M13 metalloprotease Zmp1 open states.
Authors: Wenguang G Liang / Jordan M Mancl / Minglei Zhao / Wei-Jen Tang /
Abstract: Zinc metalloprotease 1 (Zmp1), a Mycobacterium tuberculosis 75 kDa secreted enzyme, mediates key stages of tuberculosis disease progression. The biological activity of Zmp1 presumably stems from its ...Zinc metalloprotease 1 (Zmp1), a Mycobacterium tuberculosis 75 kDa secreted enzyme, mediates key stages of tuberculosis disease progression. The biological activity of Zmp1 presumably stems from its ability to degrade bacterium- and/or host-derived peptides. The crystal structures of Zmp1 and related M13 metalloproteases, such as neprilysin and endothelin-converting enzyme-1 were determined only in the closed conformation, which cannot capture substrates or release proteolytic products. Thus, the mechanisms of substrate binding and selectivity remain elusive. Here we report two open-state cryo-EM structures of Zmp1, revealed by our SAXS analysis to be the dominant states in solution. Our structural analyses reveal how ligand binding induces a conformational switch in four linker regions to drive the rigid body motion of the D1 and D2 domains, which form the sizable catalytic chamber. Furthermore, they offer insights into the catalytic cycle and mechanism of substrate recognition of M13 metalloproteases for future therapeutic innovations.
History
DepositionJun 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 14, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.title / _citation.year
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Probable zinc metalloprotease Zmp1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,1982
Polymers78,1331
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area100 Å2
ΔGint-26 kcal/mol
Surface area27510 Å2

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Components

#1: Protein Probable zinc metalloprotease Zmp1


Mass: 78132.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: zmp1, Rv0198c / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: I6X8R2
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MYCOBACTERIUM TUBERCULOSIS ZINC METALLOPROTEASE / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 6.8
Details: 20 mM HEPES (pH 6.8), 150 mM NaCl, 0.5 mM bMe, 20 mM Trimethylamine N-oxide dihydrate
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 308 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
4CTFFINDCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 541716 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0055241
ELECTRON MICROSCOPYf_angle_d0.6527137
ELECTRON MICROSCOPYf_dihedral_angle_d10.556720
ELECTRON MICROSCOPYf_chiral_restr0.045754
ELECTRON MICROSCOPYf_plane_restr0.005951

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